Proper differentiation of the intestinal epithelium underlies nearly all aspects of intestinal health. For example, improper epithelial barrier function is a critical contributor to inflammatory bowel diseases, and improper differentiation leads to poor terminal digestion and nutrient absorption, and increases susceptibility to cancers. Transcription factors that promote differentiation in the intestinal epithelium are known, but a gap exists in understanding how these factors interact with the transcriptional machinery to drive intestinal differentiation. New technologies now make it feasible to measure chromatin looping events and RNA Polymerase kinetics in vivo. These processes are unexplored in the intestine and likely to have intestine-specific mechanisms. The long term goal of our work is to resolve and manipulate the mechanisms of intestinal differentiation; our central hypothesis is that unexplored transcriptional mechanisms are dynamically regulated as cells differentiate in the intestine. These mechanisms include: 1) enhancer-promoter chromatin looping, 2) RNA polymerase loading, and 3) transcriptional elongation. The proposal will also examine how the pro- differentiation BMP/SMAD4 signaling pathway intersects with transcription factors to drive differentiation. A new, compound knockout of both HNF4A and HNF4G transcription factors leads to severe disruption of intestinal differentiation. ChIP-seq and RNA-seq identify hundreds of genes that are activated upon villus differentiation and depend upon HNF4 for expression. This novel transgenic model provides a rigorous system to apply new technologies and define intestinal regulatory mechanisms driving differentiation. The rationale of this proposal is to discover intestine-specific transcriptional regulatory mechanisms that can be exploited for the gain of human health. The novel transgenic mouse will be leveraged to define regulatory mechanisms required to activate differentiation genes in vivo. Aim 1 will apply new technologies to tackle unexplored transcriptional mechanisms driving differentiation. Aim 2 will explore a new paradigm for intestinal differentiation through the coordinated actions of BMP signaling and HNF4 factors. We expect to identify new mechanisms through which differentiation of enterocytes is achieved. Aims 1 & 2 are significant in that they can help us understand the impacts of genetic variants at enhancers, identify novel co-regulators, and lead to therapeutics that alter the homeostatic balance of intestinal differentiation that are relevant for treatments of IBD, cancer, and in regenerative medicine. HNF4 and SMAD4 are both strongly implicated in human IBD and Colon Cancer. The proposed research is innovative in the use of novel transgenic models, state-of-the-art epigenomics approaches, and human organoid cultures. Innovative ideas include the new paradigm of differentiation control by concerted efforts of HNF4 and BMP signaling, and provide a new concept through which the field can move forward to find therapeutic opportunities to control epithelial differentiation.